Cryopreservation is a process used to stabilize biological materials at very low temperatures. Previous attempts to freeze biological materials, such as living cells often results in a significant loss of cell viability and in some cases as much as 80% or more loss of cell activity and viability.
Cell damage during cryopreservation usually occurs as a result of intracellular ice formation within the living cell during the freezing step or during subsequent recrystallization. Rapid cooling often leads to formation of more intracellular ice since water molecules are not fully migrated out of the cell during the short timeframe associated with the rapid cool-down rates. Intercellular ice formation also can arise during recrystallization that occurs during the warming or thawing cycles. If too much water remains inside the living cell, damage due to initial ice crystal formation during the rapid cooling phase and subsequent recrystallization during warming phases can occur and such damage is usually lethal.
On the other hand, slow cooling profiles during cryopreservation often results in an increase in the solute effects where excess water is migrated out of the cells. Excess water migrating out of the cells adversely affects the cells due to an increase in osmotic imbalance. Thus, cell damage occurs as a result of osmotic imbalances which can be detrimental to cell survival and ultimately lead to cell damage and a loss of cell viability.
Current cryopreservation techniques involve using either conductive based cryogenic cooling equipment such as a cold shelf or lyophilizer type freezer unit or convective based cryogenic cooling equipment such as controlled rate freezers and cryo-cooler units. Such equipment, however, is only suitable for relatively small volume capacities and not suitable for commercial scale production and preservation of biological materials such as therapeutic cell lines. For example, the largest commercially available controlled rate freezer suitable for use with biological materials holds only about 8000 or so closely packed vials. One such system is the Kryo 1060-380 capable of storing 8000×2 ml ampoules. Such existing controlled rate freezers, including the Kryo 1060 series, also suffer from the non-uniformity in cooling vial to vial due, in part, to the non-uniform flow of cryogen within the freezers and the requirement for close packing of the vials within the freezer. The size of individual conventional freezers is limited due to these non-uniform effects. As conventional controlled rate freezers are scaled up in size the non-uniformities in cooling increase. Consequently, the size of conventional controlled rate freezers must be limited to prevent non-uniform sample-to-sample properties due to non-uniform cooling of each sample. The only effective way to further increase the quantity of samples processed at once using conventional controlled rate freezers is to use multiple controlled rate freezers.
Many conventional freezing systems utilize internal fans to disperse cryogen around the unit and deliver the refrigeration to the vials via convection. Such convection based cooling or freezing systems cannot achieve temperature uniformity as the vials are often located at various distances from the internal fan or packed in the shadow of other vials or trays. Vials of biological material exposed to high velocity turbulent flow of cryogen are typically cooled at a different rate and often much faster than vials situated further away from the fan.
There are also existing lyophilizer type of control rate freezers that can handle large volumes of vials but typically rely on thermal conduction between cold shelves in the lyophilizer unit and the vials. However, it is impossible to provide a uniform conductive surface area on the bottom of each glass vial since most glass vial bottoms are concave. Therefore, temperature variations during the freezing process from vial to vial are the biggest drawback for these types of equipment. Furthermore, the cooling rate can be painfully slow due to the very small conductive surface of the vial that remains in contact with the cold shelves.
Prior attempts to mitigate the loss of cell activity and viability involved the use of cryoprotective additives such as DSMO and glycerol. Use of such cryoprotectives during the cryopreservation process has demonstrated a reduction in cell losses attributable to the freezing and subsequent thawing cycles. However, many cryoprotectants such as DSMO are toxic to human cells and are otherwise not suitable for use in whole cell therapies. Disadvantageously, cryoprotectants also add a degree of complexity and associated cost to the cell production and preservation process. Also, cryoprotectants alone, have not eradicated the problem of loss of cell activity and viability.
Another problem associated with the above mentioned prior art systems is a lack of control with respect to the uniformity of the nucleation temperature between the multiple vials. This variability in the nucleation temperature of the multiple vials can lead to non-uniform vial-to-vial properties. Such properties can include cell activity and viability as well as the crystal structure of the frozen material and the time needed to complete a freeze drying process. Consequently, controlling the generally random process of nucleation in the freezing stage of a cryopreservation, lyophilization, or freeze-drying process to increase the product uniformity from vial-to-vial in the finished product would be highly desirable in the art.
In a typical pharmaceutical freeze-drying process, multiple vials containing a common aqueous solution are placed on shelves that are cooled, generally at a controlled rate, to low temperatures. The aqueous solution in each vial is cooled below the thermodynamic freezing temperature of the solution and remains in a sub-cooled metastable liquid state until nucleation occurs.
The range of nucleation temperatures across the vials is distributed randomly between a temperature near the thermodynamic freezing temperature and some value significantly (e.g., up to about 30° C.) lower than the thermodynamic freezing temperature. This distribution of nucleation temperatures causes vial-to-vial variation in ice crystal structure and ultimately the physical properties of the lyophilized product. Furthermore, the drying stage of the freeze-drying process must be excessively long to accommodate the range of ice crystal sizes and structures produced by the natural stochastic nucleation phenomenon.
Additives have been used to increase the nucleation temperature of sub-cooled solutions. These additives can take many forms. It is well known that certain bacteria (e.g., Pseudomonas syringae) synthesize proteins that help nucleate ice formation in sub-cooled aqueous solutions. Either the bacteria or their isolated proteins can be added to solutions to increase the nucleation temperature. Several inorganic additives also demonstrate a nucleating effect; the most common such additive is silver iodide, AgI. In general, any additive or contaminant has the potential to serve as a nucleating agent. For instance, lyophilization vials prepared in environments containing high particulate levels will generally nucleate and freeze at a lower degree of sub-cooling than vials prepared in low particulate environments.
All the nucleating agents described above are labeled “additives,” because they change the composition of the medium in which they nucleate a phase transition. These additives are not typically acceptable or desirable for FDA regulated and approved freeze-dried pharmaceutical products. These additives also do not provide control over the time and temperature when the vials nucleate and freeze. Rather, the additives only operate to increase the average nucleation temperature of the vials.
Ice crystals can themselves act as nucleating agents for ice formation in sub-cooled aqueous solutions. In the “ice fog” method, a humid freeze-dryer is filled with a cold gas to produce a vapor suspension of small ice particles. The ice particles are transported into the vials and initiate nucleation when they contact the fluid interface.
The “ice fog” method does not control the nucleation of multiple vials simultaneously at a controlled time and temperature. In other words, the nucleation event does not occur concurrently or instantaneously within all vials upon introduction of the cold vapor into the freeze-dryer. The ice crystals will take some time to work their way into each of the vials to initiate nucleation, and transport times are likely to be different for vials in different locations within the freeze-dryer. For large scale industrial freeze-dryers, implementation of the “ice fog” method would require system design changes as internal convection devices may be required to assist a more uniform distribution of the “ice fog” throughout the freeze-dryer. When the freeze-dryer shelves are continually cooled, the time difference between when the first vial freezes and the last vial freezes creates a difference in the temperature between vials, which will also increase the vial-to-vial non-uniformity in the final freeze-dried products.
Vial pre-treatment by scoring, scratching, or roughening has also been used to lower the degree of sub-cooling required for nucleation. As with the other prior art methods, vial pre-treatment also does not impart any degree of control over the time and temperature when the individual vials nucleate and freeze, but instead only increases the average nucleation temperature of all vials.
Vibration has also been used to nucleate a phase transition in a metastable material. Vibration sufficient to induce nucleation occurs at frequencies above 10 kHz and can be produced using a variety of equipment. Often vibrations in this frequency range are termed “ultrasonic,” although frequencies in the range 10 kHz to 20 kHz are typically within the audible range of humans. Ultrasonic vibration often produces cavitation, or the formation of small gas bubbles, in a sub-cooled solution. In the transient or inertial cavitation regime, the gas bubbles rapidly grow and collapse, causing very high localized pressure and temperature fluctuations. The ability of ultrasonic vibration to induce nucleation in a metastable material is often attributed to the disturbances caused by transient cavitation. The other cavitation regime, termed stable or non-inertial, is characterized by bubbles that exhibit stable volume or shape oscillations without collapse. U.S. Patent Application 20020031577 A1 discloses that ultrasonic vibration can induce nucleation even in the stable cavitation regime, but no explanation of the phenomenon is offered. GB Patent Application 2400901A also discloses that the likelihood of causing cavitation, and hence nucleation, in a solution using vibrations with frequencies above 10 kHz may be increased by reducing the ambient pressure around the solution or dissolving a volatile fluid in the solution.
An electrofreezing method has also been used in the past to induce nucleation in sub-cooled liquids. Electrofreezing is generally accomplished by delivering relatively high electric fields (1 V/nm) in a continuous or pulsed manner between narrowly spaced electrodes immersed in a sub-cooled liquid or solution. Drawbacks associated with an electrofreezing process in typical lyophilization applications include the relative complexity and cost to implement and maintain, particularly for lyophilization applications using multiple vials or containers. Also, electrofreezing cannot be directly applied to solutions containing ionic species (e.g., NaCl).
Recently, there have been studies that examined the concept of “vacuum-induced surface freezing” (See e.g., U.S. Pat. No. 6,684,524). In such “vacuum induced surface freezing”, vials containing an aqueous solution are loaded on a temperature controlled shelf in a freeze-dryer and held initially at about 10 degrees Celsius. The freeze-drying chamber is then evacuated to near vacuum pressure (e.g., 1 mbar) which causes surface freezing of the aqueous solutions to depths of a few millimeters. Subsequent release of vacuum and decrease of shelf temperature below the solution freezing point allows growth of ice crystals from the pre-frozen surface layer through the remainder of the solution. A major drawback for implementing this ‘vacuum induced surface freezing’ process in a typical lyophilization application is the high risk of violently boiling or out-gassing the solution under stated conditions.
Improved control of the nucleation process could enable the freezing of all unfrozen solution containers in a cryogenic chiller or freeze-dryer to occur within a narrower temperature and time range, thereby yielding a product with greater uniformity from sample-to-sample. With regards to freeze-drying systems, controlling the minimum nucleation temperature affects the ice crystal structure formed within the vial and allows for a greatly accelerated freeze-drying process.
In view of the above, what is needed is a method and system to control the uniformity of the temperature profiles and nucleation temperatures of the multiple containers so as to provide a more uniform finished product sample-to-sample. Moreover, the system and method should be both efficient and readily scalable to handle commercial scale production.